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. 2012 Mar 1;23(5):834–852. doi: 10.1091/mbc.E11-08-0731

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© 2012 Yi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Characterization of TCR MC dynamics at the LP/dSMAC and LM/pSMAC regions of the IS. (A) Time-lapse images showing TCR MC movements in the LP/dSMAC and LM/pSMAC regions of the IS in a representative Jurkat cell stimulated on a planar bilayer (see also Supplemental Movie S6). TCR MCs (pseudocolored green) reported by rhodamine X–labeled anti-CD3ε antibody in the bilayer. F-Actin (pseudocolored red) reported by GFP–F-tractin-P. White arrowheads point to a TCR MC undergoing inward movement. The frame at 40 s that is boxed in orange indicates the moment when this TCR MC moved from the LP/dSMAC into the LM/pSMAC during its centripetal movement. (B1) Kymograph of GFP–F-tractin-P in the region corresponding to the yellow line in A (0 s), (B2) kymograph of a TCR MC in the region corresponding to the yellow line in A (0 s), and (B3) merged image between B1 (red) and B2 (green). The positions of the LP/dSMAC and LM/pSMAC regions of the IS are indicated by the brackets above B1–B3. The boundary between the LP/dSMAC and LM/pSMAC is indicated by the orange line in B1–B3. The time scale of the kymographs in B1–B3 is indicated to the left of B1. (C) Paths of all visible TCR MCs in a representative Jurkat cell stimulated on a planar bilayer. TCR MC paths in the LP/dSMAC are shown in red, and TCR MC paths in the LM/pSMAC are shown in green. The paths are discontinuous because we purposely avoided making measurements at the dSMAC/pSMAC boundary. (D) Spatial and kinetic relationship between a TCR MC and surrounding actomyosin II arcs in the LM/pSMAC region of the IS (see also Supplemental Movie S7). (D1) GFP–myosin IIA HC (pseudocolored red) and TCR MCs (pseudocolored green) at the IS of a Jurkat cell stimulated on a planar bilayer. (D2) Magnified image of the boxed region (white) in D1. (D3) Relative intensities for myosin IIA (red) and a TCR MC (green) across the yellow line in D2. (D4) Kymograph of two prominent myosin IIA-containing arcs in the region corresponding to the yellow line in D1, (D5) kymograph of a TCR MC in the region corresponding to the yellow line in D1, and (D6) merged image between D4 (red) and D5 (green). The time scale of the kymographs in D4–D6 is indicated to the right of D6. Scale bars in A, B3, and D6, 2 μm; in D1, 5 μm.